PGIMER research updates. AMR at human animal interface: Taneja. Dr. Neelam .2017

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1 AMR at human animal interface: PGIMER research updates.2017 Dr. Neelam Taneja Professor and In-charge Enteric Laboratory Postgraduate Institute of Medical Education and Research Chandigarh

2 PGIM MER 000 bed tertiary care referral centre in handigarh, North India aters to patients from seven adjoining tates (Chandigarh, Punjab, Haryana, imachal Pradesh, Jammu and Kashmir, estern parts of Uttar Pradesh, ttaranchal, and some parts of Rajasthan) hus representing a large geographical rea,42,3501 outpatients and dmission in

3 Enteri ic Lab iagnostic(conventional as well Urine and Stool samples Load /year NABL accreditation as molecular) urveillance and referral servi ices investigating and managin utbreaks of cholera, and food poisoning in this geographic area Research in the field of diarr rhea, food borne infections an rinary tract infections with special focus on epidemiology an rug resistance and pathogenes sis.2017

4 ntimicrobial resistance in Ente eric and Uro pathogens is bein tudied at community and hospital level by both phenotypic an olecular assays onventional Culture for Salmonella, Shigella,Vibrio cholerae Campylobacter,STEC and Yersinia,C.difficle ntigen detection for STEC, Campylobacter,C difficle olecular tests Multiplex PCR for diarrhoeagenic E.coli Shigella PCR STEC PCR Campylobacter PCR apid dipstick for cholera and dysentery.2017

5 Food borne Pathogens Vibrios, Bacillus cereus, Shigella,Aeromonas, Pleisomonas, Yersinia Campylobacter, Staphyloccocus au ureus, Listeria, testing of milk samples by total plate counts, methylene blue test, turbidity test, coliform tests fo milk, full water testing for coliforms, E coli, Enterococci, V. cholerae testing of water directly for diarrhoeagenic E.coli using in house protocols (PCR). Molecular assays to detect directly from food samples Special food (RUTF) which was developed to treat malnutrition was tested ed by us for component analysis a s and shelf life of the prepared ed food Receive request from Government labs for testing commercial food and water samples 2017

6 apacity for molecular epidemiology AFLP PFGE MLVA Ribotyping Plasmid typing Rep-PCR and RAPD MLST WGS.2017

7 Public Health Contribution lic health role and surveillance nvestigated 25 outbreaks of cholera and gastroenteritis. Visit the area and collect water samples, sto ol samples and study the factors responsible holera. ue to fast identification of source of wate er contamination, we could control the chol utbreaks in a week. e are also studying the molecular l epid demiology of V. cholerae causing the rec pidemics in our region byaflp, ribotyping and PFGE. 2017

8 HO APW project and DFC engthening gdistrict public health laboratories for laboratory surveillance of communicable diseases of break potential of phase 1 o assist in capacity building of the district laboratories. Punjab Moga, Bhatinda, Hoshiarpur,Sangrur Haryana Ambala, Faridabad, Bhiwani, Panchkula Uttarakhand Dehradun, Haridwar, Tehri Garhwal,Pauri o assist in technical operations & provision of resource es needed to ensure required quality of laboratory rocedures. o formulate a framework for establishment of quality control & quality assessment schemes in these aboratories. o periodically visit the laboratories at district level & monitor the progress

9 HO APW project and DFC-Phase 2 Stool referral system :A total of 336 stool samples were colle ected and submitted from the following centers Moga (N=58, Ambala (N=38), Sangrur (N=17), Panchkula (N=72) and Pauri (N=32) DPHLs. These samples were collected from PHC and DPHL level by active surveillance Most of the samples (231, 68%) were sent by local couriers and 95% were received in proper conditions, though some were not sent in double packaging Outbreaks of cholera were reported from Ambala, Moga and Panchkula,samples were also submitted from suspected cases from Sangrur, Uttarakhand and Chandigarh for confir rmation. Fourteen outbreaks of diarrhoea / food poisoning occurred in 2010 and 2011 followed by 4 outbreaks each of dengue chickenpox, measles and viral fever, 3 of jaundice, 2 of typhoid one each of viral encephalitis and rubella. Tool for assessment of biosafety in laboratory The culture facilities were established at the following centres Ambala, Bhiwani, Haridwar, Pauri. Quality assurance panels were sent and responded

10 Food borne illnesses Foodborne illness are of serious concern to public health h worldwide 90% bacterial infections followed by parasitic and viral Major bacterial pathogens : Campylobacter, Salmonella, Diarrhoeagenic coli, Listeria, Yersinia, Shigella, Vibrio, etc. Salmonella is the leading cause of Alert;2008) death followed by Campylobacter (C Both occur in intestinal tract of sheep p, goat, pigs and poultry

11 E. coli is commensal microbiota but some may be pathogenic strain i.e. EPEC, ETEC,EAEC,EIEC,STEC Transfer of virulence genes can occur through lateralgene transfer Diarrhoeagenic E. coli caused maxi imum hospitalizations Contaminated food and water are main sources of infections

12 Indian scenario Burden of foodborne diseases is un nknown No surveillance system No national database of epidemiology of common food borne pathogens Foodborne infections occur as sporadic cases and also as outbreaks Underreporting of foodborne outbreaks and cause is rarely established. Foodborne gastroenteritis is clubbed with acute diarrhoea and is not notifiable

13 In our region too foodborne illn nesses are very common but no investigated or reported In a study conducted by us, in collaboration with WHO, we found tha every year 1,400 to 31,000 cases of suspected food and water borne infections were being reported at the district public health lab (DPHLs) across Punjab, Haryana and Uttarakhand Every year one to three outbreaks of food poisoning reportedly occurred at the DPHLs (Unpublishe ed data PGIMER) Almost all of these go uninvestigated

14 How antibiotics in livestock affect us used for prophylactic, therapeutic and g rowth promotion suppress gut flora leaving more nutrients to be absorbed by animal leading to greater gain in weight Most of the antibiotic classes are the ones used for humans Overuse has lead to emergence of antib iotic resistance Residues of these antibiotics remain active in animals for certain time and are also excreted in faeces Consumption of trace levels of these residues in foods may alter human intestina microflora and cause resistance gene transfer Low levels of abs released in environment accumulate and affect bacteria and cause transfer of resistance genes 017

15 Global antimicrobial consumption in livestock (Van Bockerel; 2015)

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17 What we lack in India Guidelines on antibioticusein i feed are available, but not implemente Antibiotics used for human dise ase treatment are used in growt promoters No monitoring of residue limits in food meat in Indian markets We do not know the antibiotic resistance pattern or resistance gen pool in food animals Weareunawareoftherateoftransmission of antibiotic resistance t humans

18 Antibiotics Class / active compound Furazolidone Nitrofuran Human + veterinary ly imp Bacitracin Peptides from B. Human (topical) + licheniformis veterinary tics Neomycin + doxycycline Aminoglycoside + Human + veterinary stin tetracycline Colistin Polymyxin E Human + veterinary floxacin Amoxicillin Penicillin Human + veterinary mycin Levofloxacin Fluoroquinolone Human + veterinary Colistin + doxycycline Polymyxin E + tetracycline Human + veterinary loxacin kacin xcillin Neomycin + doxycycline Aminoglycoside + tetracycline Human + veterinary Neomycin + oxytetracycline Aminoglycoside + tetracycline Human + veterinary Neomycin + Aminoglycoside id + sulphadizamine sulphonamide Avilamycin Orthosomycin Oxytetracycline Tetracycline Human + veterinary hly imp halquinol chloroxine ibiotics Tilmicosin macrolide veterinary Perfloxacin quinolone Discontinued in human Used in Veterinary Tiamulin Pleuromutilin (inhibit Veterinary roribosomes protein synthesis by binding to 50S subunit of ) cycline Oxyclozanide salicylanilide anthelmintic Veterinary tetracycline ycycline 017 Enrofloxacin Chlorotetracycline Ceftiofur Amikacin Tylosin Flumequine Fluroquinolone Tetracycline Third generation cephalosporin Aminoglycoside Macrolide Fluoroquinolone Veterinary Veterinary Veterinary + human Veterinary Discontinued in human Used in Veterinary

19 One Health Approach 017

20 WHO AGIS SAR Project Monitoring i the Antimicro i obial resistance profile of bacterial food borne pathogens in Humans, food animals and retail meat in India 017

21 uman Samples ample collection Total samples collected = 1968 proforma for history was filled Diarrheal stool samples were colle cted & transported in Cary and Blair transport mediumm

22 Ludhiana Kangra Hamirpur Shimla Map showing sites of human sto sample collection PGIMER Ambala Manimajra Dehradun Rishikesh Panchkula Haridwar Haldwani Rudrapur Jaipur

23 2. Sample Processing Stool sample in Cary and Blair medium E. coli Salmonella Campylobacter MacConkey agar + Peptone broth 37 C for overnight incubation MacConkey agar + XLT4 agar + CHROMagar (37 C ) + Rappaport broth (42 C ) for overnight incubation Campy Cefex agar + CHROMagar + Bolton broth at 42 C under microearophillic conditions for 48 hours Subculture from Peptone to MacConkey Subculture from Rappaport broth to agar MacConkey agar + XLT4 37 C for overnight incubation 37 C foroverni overni ight incubation Subcullture from Bolton broth to Campy Cefex agar + CHROMagar Lactose fermenting and Non lactose fermenting colonies NLF colonies on MacConkey agar Pink colonies with black centre on XLT4 Greyish colonies + oxidase positive Camy cefex agar Dark Pink colonies on CHROMaga Colonies were identified by MALDI TOF * Salmonella identified up to genus level Further serotyping to be done

24 nimal samples ples collected: 839 l: 487 t: 352

25 Sh i i f p Showing sites of mal sample collection

26 Animal samples Animal (goat, sheep, pig) meat and stool samples were collected from slaughter house, Chandigarh which h receives animals from Chandigarh and nearby areas of Panchkula, Manimajra, Mohali and Punjab Poultry stool samples were collected from farms in and around Chandigarh These samples were processed for Campylobacter, Non Typhoida Salmonella and E. coli detection by culture methods 017

27 imal sample processing Sheep/ /Goat, Pig, Poultry Stool Meat Direct inoculation on respective agar + Enrichment broth for E. coli, Salmonella, Campylobacter Approx. 10 g of meat in 990 ml Buffered peptone water+ Rappaport broth + Bolton broth Subcultured on respective agar for E.col Salmonella, Campylobacter

28 Table showing compiled results from all animals urce Sample (n) Campylobacter (%) Salmonella (%) E. coli (%) at Stool (90) 11 MALDI (12.22) identified 7 (7.78) 67 (74.44) Multiplex PCR Meat (191) speciess 7 (3.66) 3 (1.57) identified 128 (67.02) pathotypes Campyloba cter g Stool (57) 21 (36.84) jejuni (38.79%) 22 (38.6) 36 EAEC (63.16) (1%) Meat (151) 42 (27.81) Campylobacter coli (58.41%) 29 (19.21) 116 (76.82) EPEC (8%) icken Stool (340) 131 (38.53) 38 (11.18) 298 (87.65) STEC/EHEC (4.5%) Meat (10) 2 (20) Campylobacter hyointestinalis 0 10 (100) tal (2.33%) (25.48) 99 (11.8) 655 ETEC (78.07%) (5.5%) 17 5% samples has both Campylobacter and Salmonella

29 Table showing results of human stool samples te Locations Number of Campyl obacter Salmonella DEC E. coli Pathotypes samples ndigarh PGIMER yana Panchkula 35 4 Manimajra Ambala 29 2 achal Pradesh Kangra 8 0 Shimla 70 3 Hamirpur 11 1 arakhand Rudrapur 19 3 Haldwani 13 0 Rishikesh h 86 4 Dehradun 4 0 Haridwar 38 3 asthan Jaipur 24 3 jab Ludhiana 8 0 EAEC EPEC ETEC al (2.32%) 24 (1.24%) 98 (5.05%) 83 (4.28%) 94 (4.84%

30 Antibiotic usage and residues 017

31 Antibiotic susceptibility 017

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33 72% ESBL producers High frequency of resistance to fluroquinolones Resistance seen against 3 rd generation cephalosporins No resistance to carbapenems

34 istance to fluroquinolones Low resistance was detected

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36 017

37 Antimicrobial i susceptibility in commensal le. coli lifrom meat samples Percentage R MS S 20 0 AM AK GM CIP LVX ETP TZP IPM FEP CTR FOX COT TE CL 017

38 MDR Commensal E. coli ESBL producing E. coli 23% 16% 61% 32% chicken goat pig 30% chicken goat pig 38% 017

39 Antibiotic resistance genes 017

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41 MLST of ET TEC isolates One of the ETEC strain belongs to ST131 complex and so ome new sequence types have been identified ST131 has emerged worldwide and is linked to bla CTX M M 15. Extensive studies investigating the association of th multilocus sequence typing (MLST) clonal complex ST131 and bla CTX M 15 have been reported from Canada, India, Kuwait, France, Switzerland, Portugal, Spain, Korea and Japan; worldwide dissemination of bla CTX M seems to be linked to this clonal complex, which is situatedinthephylogeneticgroupb2.this demonstratesth need for constant surveillance in developing countries to prevent the spread of these multiresistant isolates Another ETEC strain belong to ST 117strains isolated worldwide (such as Brazil, USA, Egypt, Denmark, S Lanka, and South Korea) have ColV related plasmids, were involved in osteomyelitis and arthritis cases These STs are commonly shared by APEC and human ExPEC strains These hypothetical hybrid strains could have the potential to infect humans and birds. Many new STs are found 017

42 Serotyping of Salmonella isolates Future work Perform MIC testing of Campylobacter, Salmonella and E. col pathotypes isolates using sensititree plates Estimate Antibiotic residue levels in meat samples by LC MS MS Farm enviorns, water sewage using one health approach Perform Whole Genome Sequencing of isolates to understand the transmission dynamics and determine AMR with more precision 017

43 Team Members 017 Dr. Balvinder Mohan, Dr. Chandra Deo, Harpreet, Naveen, Vinay Modgil, Jaspreet Mahindroo, Vishal, Varun Shahi, Meenakshi, Yousuf, Pinki Shankar, Ritu Verma, Bhaskar Samaui, Dhananjay

44 cknoweldgements Dr Awa Adriana Kane (WHO AGISA AR)for funding Dr Sidharth Thakur, Dr Paula, Dr Ruby Network partners Dr Bhavneet and Dr Surjit Singh My lab and team mates ICMR for funding HOD Medical Microbiology for his support